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WO2003104270A2 - Genes de la duduline 2, produits d'expression, modele animal non humain : utilisations dans les maladies hematologiques humaines - Google Patents

Genes de la duduline 2, produits d'expression, modele animal non humain : utilisations dans les maladies hematologiques humaines Download PDF

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WO2003104270A2
WO2003104270A2 PCT/EP2003/005920 EP0305920W WO03104270A2 WO 2003104270 A2 WO2003104270 A2 WO 2003104270A2 EP 0305920 W EP0305920 W EP 0305920W WO 03104270 A2 WO03104270 A2 WO 03104270A2
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dudulin
seq
polypeptide
amino acid
acid sequence
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PCT/EP2003/005920
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WO2003104270A3 (fr
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Johannes Grosse
Jürgen LAUFS
Boris Schneider
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Ingenium Pharmaceuticals Ag
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Priority to AU2003242633A priority Critical patent/AU2003242633A1/en
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Publication of WO2003104270A3 publication Critical patent/WO2003104270A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a non-human hematological animal model with a mutation in the dudulin 2 gene, and to the mutated dudulin 2 gene (including both the murine and human orthologues).
  • the invention further relates to the uses of the model for diagnosis and treatment of human hematological disorders in which the dudulin 2 gene is implicated, and for manufacturing medicaments for the treatment of diseases associated with defects in the dudulin 2 gene, or in the control of its expression.
  • Eukaryotic cells are characterized by biochemical and physiological processes, which under normal conditions are extremely balanced to achieve the preservation and propagation of the cells.
  • biochemical and physiological processes When such cells are components of multicellular organisms such as vertebrates, or more particularly organisms such as mammals, the regulation of the biochemical and physiological processes involves intricate signaling pathways. Frequently, such signaling pathways are constituted of extracellular signaling proteins, cellular receptors that bind the signaling proteins and signal transducing components located within the cells.
  • Signaling proteins may be classified as endocrine effectors, paracrine effectors or autocrine effectors.
  • Endocrine effectors are signaling molecules secreted by a given organ into the circulatory system, which are then transported to a distant target organ or tissue.
  • the target cells include the receptors for the endocrine effector, and when the endocrine effector binds, a signaling cascade is induced.
  • Paracrine effectors involve secreting cells and receptor cells in close proximity to each other, for example two different classes of cells in the same tissue or organ. One class of cells secretes the paracrine effector, which then reaches the second class of cells, for example by diffusion through the extracellular fluid.
  • the second class of cells contains the receptors for the paracrine effector; binding of the effector results in induction of the signaling cascade that elicits the corresponding biochemical or physiological effect.
  • Autocrine effectors are highly analogous to paracrine effectors, except that the same cell type that secretes the autocrine effector also contains the receptor. Thus the autocrine effector binds to receptors on the same cell, or on identical neighboring cells. The binding process then elicits the characteristic biochemical or physiological effect.
  • Signaling processes may elicit a variety of effects on cells and tissues, including by way of nonlimiting example induction of cell or tissue proliferation, suppression of growth or proliferation, induction of differentiation or maturation of a cell or tissue, and suppression of differentiation or maturation of a cell or tissue.
  • pathological conditions involve dysregulation of expression of important effector proteins.
  • the dysregulation is manifested as diminished or suppressed level of synthesis and secretion of protein effectors.
  • a subject may be suspected of suffering from a condition brought on by diminished or suppressed levels of a protein effector of interest. Therefore there is a need to assay for the level of the protein effector of interest in a biological sample from such a subject, and to compare the level with that characteristic of a nonpathological condition.
  • Administration of the effector to a subject in need thereof is useful in treatment of the pathological condition. Accordingly, there is a need for a method of treatment of a pathological condition brought on by a diminished, suppressed, or in some cases elevated, level of the protein effector of interest.
  • Iron important in the formation of hemoglobin and the cytochromes, is largely absorbed in the upper part of the small intestine, mainly in the ferrous form rather than the ferric form. The rate of absorption is relatively slow. Deficiencies in iron availability are often manifested as anemias in humans (characterized by a deficiency in red blood cells). Such anemia may result as a consequence of the inability to absorb sufficient iron from the intestines to fuel sufficient production of red blood cells to replace those lost in the course of normal metabolism, or due to blood loss.
  • dudulin 2 there has been no previous demonstration of the role of dudulin 2 in hematological disorders, nor of dudulin 3.
  • RW red blood cell distribution width
  • dudulin 2 has been implicated as an inducer of apoptosis in prostate cancer cells because of its homology to a rat sequence, p-Hyde.
  • p-Hyde a rat sequence
  • This invention provides a non-human animal useful as a model of dudulin disorders in humans, particularly dudulin 2 disorders.
  • Embodiments of this invention provide a validated animal model for correlating mutations or defects in the dudulin 2 gene with human hematological diseases resulting in whole or in part from these mutations or defects in the dudulin 2 gene or the protein it encodes, or in the defective function or control of the dudulin 2 gene or protein.
  • this invention provides for the first time, an animal model that demonstrates the role of dudulin 2 in hematological disorders, and particularly those hematological disorders characterized by a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal in conjunction with an elevated red blood cell distribution width ("RDW"; see Example 1) or an elevated reticulocyte-count resulting from mutations or defects in dudulin 2.
  • RW red blood cell distribution width
  • the animal model provided herein meets the currently unfulfilled need for a validated model to study the causes and precise physiological effects of such hematological diseases and provides a means to identify antagonists and inhibitors for dudulin 2 in hematological disorders.
  • the animal of the invention carries a mutated dudulin 2 gene encoding a dudulin 2 protein with a modified amino acid sequence compared to the wild type sequence.
  • the invention also provides cell lines derived from the animal model of the present invention.
  • the present invention also relates to the use of the animal model of the invention for the study of disorders associated with deficiencies in dudulin 2 and for the study of disorders associated with deficiencies in dudulin 3.
  • the invention provides methods of diagnosis for deficiencies in dudulin 2, or the gene encoding it.
  • Another embodiment provides methods of diagnosis for deficiencies in dudulin 3, or the gene encoding dudulin 3.
  • the invention provides a method for screening of preventive or therapeutic agents of disorders and symptoms associated with dudulin 2 deficiency, using the animal model of the invention.
  • the present invention provides mutated dudulin 2 polynucleotides and polypeptides, each having a modified sequence compared to the corresponding wild type sequence, as well as vectors and cell lines for expressing the muteins (mutated proteins; expression products of mutated polynucleotides) recombinantly.
  • mutated nucleic acids and polypeptides may also be used in the diagnostic and therapeutic methods contemplated herein.
  • the nucleic acid encoding a murine dudulin 2 mutein has a point mutation at position 1228 (see, SEQ ID NO:3); another embodiment provides the corresponding mutein with a substituted amino acid residue at position 395 (see, SEQ ID NO:4).
  • nucleic acid which encodes a human dudulin 2 mutein and has a point mutation at position 1317 (see, SEQ ID NO:5), and by the corresponding mutein with a substituted amino acid residue at position 395 (see, SEQ ID NO:6).
  • the present invention further provides mutated dudulin 3 polynucleotides and polypeptides, each having a modified sequence compared to the corresponding wild type sequence, as well as vectors and cell lines for expressing the muteins (mutated proteins; expression products of mutated polynucleotides) recombinantly.
  • mutated nucleic acids and polypeptides may also be used in the diagnostic and therapeutic methods contemplated herein.
  • the nucleic acid encoding a murine dudulin 3 mutein has a point mutation at position 1096 (see SEQ ID NO.35); another embodiment provides the corresponding mutein with a substituted amino acid residue at position 366 (see SEQ ID NO:36).
  • nucleic acid which encodes a human dudulin 2 mutein and has a point mutation at position 1090 (see SEQ ID NO:37), and by the corresponding mutein with a substituted amino acid residue at position 364 (see SEQ ID NO:38).
  • dudulin 2 muteins as modulators (whether agonists or antagonists) of endogenous dudulin activity, particularly of endogenous dudulin 2 activity. Consequently we contemplate pharmaceutical compositions comprising the dudulin 2 muteins of this invention plus a pharmaceutically acceptable carrier.
  • the polynucleotides encoding them and vectors bearing said polynucleotides for the prevention, treatment or amelioration of a medical condition in a mammalian subject, particularly a human subject, and in particular their use for the manufacture of a medicament for prevention, treatment or amelioration of any medical conditions characterized by a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal, an elevated reticulocyte-count, an elevated red blood cell distribution width, or a combination of these features.
  • dudulin 2 wild type polypeptides see, e.g. SEQ ID NO: 2 or 8) or dudulin 2 wild type polypeptides encoded by SEQ ID NO:9 or SEQ ID NO: 10 as modulators (whether agonists or antagonists) of endogenous dudulin activity, particularly of endogenous dudulin 2 activity. Consequently we contemplate pharmaceutical compositions comprising the dudulin 2 wild type polypeptides of this invention plus a pharmaceutically acceptable carrier.
  • dudulin 2 wild type polypeptides the polynucleotides encoding them and vectors bearing said polynucleotides for the prevention, treatment or amelioration of a medical condition in a mammalian subject, particularly a human subject, and in particular their use for the manufacture of a medicament for prevention, treatment or amelioration of any medical conditions characterized by a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal, an elevated reticulocyte- count, an elevated red blood cell distribution width, or a combination of these features.
  • dudulin 3 muteins as modulators (whether agonists or antagonists) of endogenous dudulin activity, particularly of endogenous dudulin 3 activity. Consequently we contemplate pharmaceutical compositions comprising the dudulin 3 muteins of this invention plus a pharmaceutically acceptable carrier.
  • the polynucleotides encoding them and vectors bearing said polynucleotides for the prevention, treatment or amelioration of a medical condition in a mammalian subject, particularly a human subject, and in particular their use for the manufacture of a medicament for prevention, treatment or amelioration of any medical conditions characterized by a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal, an elevated reticulocyte-count, an elevated red blood cell distribution width, or a combination of these features.
  • dudulin 3 wild type polypeptides see, e.g. SEQ ID NO:32 or SEQ ID NO:34
  • modulators whether agonists or antagonists
  • compositions comprising the dudulin 3 wild type polypeptides of this invention plus a pharmaceutically acceptable carrier.
  • the polynucleotides encoding them and vectors bearing said polynucleotides for the prevention, treatment or amelioration of a medical condition in a mammalian subject, particularly a human subject, and in particular their use for the manufacture of a medicament for prevention, treatment or amelioration of any medical conditions characterized by a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal, an elevated reticulocyte-count, an elevated red blood cell distribution width, or a combination of these features.
  • Fig. 1 is a chart diagramming the F3 -production (Fig. 1 A) and the outcross breeding schemes (Fig. IB, IC) used to map the mutation associated with observed hematological phenotype abnormalities to mouse chromosome 1.
  • the parallel upright symbols represent the two alleles of the genome, cross bars represent mutation events; thick upright symbols represent the wild type of a different mouse strain used for outcrossing.
  • m WT indicates male wild type
  • f WT indicates female wild type;
  • RF1 indicates recessive FI x FI
  • RBS indicates recessive brother-sister
  • ROC indicates recessive out-cross
  • RIC indicates recessive inter-cross
  • Abbreviations in minuscules indicate the animals involved in each breeding stage, their names indicating the stages from which they were generated.
  • Fig. 2 presents a cluster analysis of hematological data obtained from outcrossed F5 progeny of the mouse line of the invention, showing the distinction between progeny that are affected and progeny that are unaffected by the phenotype associated with the mutation. These data, clustered within two groups of approximately 25% and 75% of the total, demonstrate the presence of a recessive inherited trait according to the Mendelian rules of genetic distribution. (Example 4A provides further information regarding these data: 44 affected animals and 128 unaffected; a ratio of 25.5% to 74.5%.
  • the terms RDW and MCH are defined in
  • Example 1 • Fig. 3 shows a plot of pooled genomic DNA scores of phenotype-affected F5 mice from comparison against heterozygote F4 animals, for chromosome 1. (Further details are provided in Example 4B).
  • Fig. 4 shows an alignment of the murine and human dudulin 2 protein sequences (wild type), indicating the sequence homology between the two proteins. The position corresponding to the mutation of embodiments of the invention is underlined and highlighted,
  • Fig. 5 shows an alignment of the murine and human dudulin-family protein sequences (wild type), indicating the relation of the proteins and the conservation throughout the family of the mutated glutamine that is mutated in embodiments of the invention. This residue is underlined and highlighted in the figure.
  • the mouse dudulin 2 sequence is the corrected sequence disclosed herein as SEQ ID NO:2. The alignment was performed using CLUSTAL W (Astra Draco AB, Lund, Sweden: http://circinus.ebi.ac.uk:6543/cgi-bin/clustalw.cgi).
  • Fig. 6 shows the percentage of reticulocytes compared to all blood cells of dudulin 2 mutated mice of the invention. Animals homozygous for the mutation (mut) have an elevated reticulocyte count compared to animals heterozygous (hz) or wild type (wt) for the mutation. (See Example 2).
  • Fig. 7 shows syntheny between mouse and human concerning the chromosomal region (A) and exon intron structure (B) of murine and human dudulin 2.
  • A chromosomal region
  • B exon intron structure
  • the dudulin 2 gene is represented by the horizontal line, vertical bars represent exons, and the numbers above and below the line indicate the sizes in base pairs of, respectively, the exons (above) and the introns (below).
  • Fig. 8 shows an aligned murine dudulin 2 protein sequences, indicating the differences between the following proteins: 'C indicates dudulin 2 of mouse strains
  • the present invention provides nucleic acid sequences encoding wild type and mutated murine and human dudulin 2 as well as nucleic acid sequences of novel murine and human dudulin 2 family members (SEQ ID NO:31 and SEQ ID NO:33, respectively).
  • this invention provides a corrected nucleic acid sequence for wild type murine dudulin 2 (SEQ ID NO:l; the prior art sequence in GenBank, Accession Number AY029586 is incorrect - we have presented that sequence here as SEQ ID NO:9).
  • the wild type murine dudulin 2 nucleic acid sequence is shown in SEQ ID NO:l.
  • this invention provides mutated nucleic acid sequences for murine and human dudulin 2.
  • SNP single nucleotide polymorphism
  • SEQ ID NO:3 A human nucleic acid sequence, containing the reading frame for the human orthologue nucleic acid sequence, with a SNP at the corresponding position (position 1317) in the sequence is shown in SEQ ID NO:5.
  • the wild type human dudulin 2 nucleic acid sequence is shown as SEQ ID NO:7.
  • dudulin-2 family members by searching the public databases for homologous sequences, as described in Example 4. All four genes are found in both mouse and man. Besides dudulin 2, the six-transmembrane epithelial antigen of the prostate (STEAP) [human: Accession No. NM_012449; mouse: Accession No. AF297098] and TNF- ⁇ -induced adipose-related protein (TIARP) [human: Accession No. XM_016074; mouse: Accession No. NM_054098] belong to this group. Additionally, we have identified a novel gene, which we have termed dudulin 3, which is the closest homologue of the three to dudulin 2. This gene was detected and identified by using the TBLASTN-algorithm to search the sequence of the mouse genome, as described in greater detail in Example 4.
  • Nucleic acid sequences of murine and human dudulin 3 predicted coding sequences are shown in SEQ ID NO:31 and SEQ ID NO:33, respectively, the amino acid sequences of the predicted expression products that are encoded by these nucleic acid sequences are shown as SEQ ID NO:32 (murine) and SEQ ID NO:34 (human).
  • Nucleic acid sequences of the mutant murine and human dudulin 3 predicted coding sequences that are specific embodiments of the present invention are shown in SEQ ID NO:35 and SEQ ID NO: 37, respectively, and the amino acid sequences of the muteins that these coding sequences express and which are also specific embodiments of the present invention are shown as SEQ ID NO:36 (murine) and SEQ ID NO:38 (human).
  • nucleic acid sequences that are contemplated as within the scope of this invention include complementary and antisense sequences: particularly sequences that hybridize to the nucleic acid sequences encoding dudulin 3 shown in SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37, other than those known in the prior art (as described above), these nucleic acid sequences of the invention having at least 75 %, preferably at least 80 %, more preferably at least 90 %, even more preferably 95% and most preferably at least 99 % sequence homology to the antisense sequence of SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37; specific embodiments of the invention that are contemplated are nucleic acid sequences that comprise the antisense sequences to SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37. Additionally, nucleic acid sequences that are degenerate with respect
  • nucleic acid sequences encoding dudulin 2 or mutant dudulin 2 of the invention may exist alone or in combination with other nucleic acids as, for example, vector molecules, such as plasmids, including expression or cloning vectors.
  • nucleic acid sequences encoding dudulin 3 or mutant dudulin 3 of the invention may exist alone or in combination with other nucleic acids as, for example, vector molecules, such as plasmids, including expression or cloning vectors.
  • nucleic acid sequence refers to any contiguous sequence series of nucleotide bases, i.e., a polynucleotide, and may be ribonucleic acids (RNA) or deoxy-ribonucleic acids (DNA).
  • RNA ribonucleic acids
  • DNA deoxy-ribonucleic acids
  • nucleic acid sequence is cDNA.
  • isolated nucleic acid molecule is one, which is separated from other nucleic acid molecules ordinarily present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences, which naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism that is the natural (wild type) source of the DNA.
  • Dudulin 2 molecules and dudulin 3 molecules can be isolated using standard hybridization and cloning techniques, as described, for instance, in Sambrook et al. (eds.), MOLECULAR CLONING: A LABORATORY MANUAL (2 nd Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, NY, 1993.
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to dudulin 2 nucleotide sequences and oligonucleotides corresponding to dudulin 3 nucleotide sequences can be prepared by standard synthetic techniques, e.g. , using an automated DNA synthesizer.
  • oligonucleotide refers to a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction.
  • a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
  • oligonucleotide is used to refer to a series of nucleotides (a polynucleotide) of about 100 nucleotides (nt) or less, e.g., portions of a nucleic acid sequence of about 100 nt, 50 nt, or 20 nt in length, preferably nucleotide sequences of about 15 nt to 30 nt in length.
  • the term “complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof.
  • a “homologous nucleic acid sequence” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by a homology at the nucleotide level or amino acid level, respectively.
  • Homologous nucleotide sequences encode those sequences coding for isoforms of dudulin 2 polypeptides. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, different genes can encode isoforms.
  • dudulin 2 and dudulin 3 sequences described herein indicates that the structures of these two protein types are similar, with many structural domains in common, and consequently the metabolic and physiological functions of dudulin 2 and dudulin 3 are related.
  • stringent hybridization conditions refers to conditions under which a probe, primer or oligonucleotide will hybridize to its target sequence, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes, primers or oligonucleotides (e.g., 10 nt to 50 nt) and at least about 60°C for longer probes, primers and oligonucleotides.
  • Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide. Stringent conditions are known to those skilled in the art and can be found in Ausubel et al.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6X SSC, 50 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65°C, followed by one or more washes in 0.2X SSC, 0.01% BSA at 50°C.
  • the present invention also provides murine and human dudulin 2 amino acid sequences of wild type polypeptides and muteins.
  • the present invention identifies certain known murine and human amino acid sequences as being dudulin 2 family members (see Example 4; Fig. 5).
  • the present invention also identifies amino acid sequences, which have been theoretically predicted from open reading frames, as being members of the dudulin 2 family, herein termed dudulin 3 (SEQ ID NO:32 and SEQ ID NO:34, for dudulin 3 of mouse and human, respectively).
  • dudulin 3 proteins are here proposed to have similar biochemical and physiological functions to those of dudulin 2, described herein, based upon the high degree of homology between the dudulin 2 and dudulin 3 amino acid sequences.
  • the wild type murine (C3H/HeJ) dudulin 2 amino acid sequence is shown as SEQ ID NO:2.
  • a mutated version wherein glutamine residue (Gin; Q) at position 395 has been mutated to a lysine residue (Lys; K) is shown in SEQ ID NO:4.
  • a preferred embodiment of a human mutein with the same amino acid change at the corresponding position (native Gin residue to mutant Lys residue at position 395) in the sequence is shown in SEQ ID NO:6.
  • the wild type human dudulin 2 amino acid sequence is shown as SEQ ID NO: 8.
  • Known murine family members of the dudulin family are murine STEAP (Accession No.: AAK83126), also known as dudulin (Accession No.: AAK50537; not shown in the alignment of Figure 5 due to errors within the published sequence) and murine TIARP (Accession No.: NP_473439), also known as dudulin 4 (Accession No.: AAK40270, not shown in the alignment of Figure 5 due to errors within the published sequence).
  • Known human family members of the dudulin family are human STEAP (Accession No.: NP_036581) and FLJ23153 (Accession No.: XP_016074).
  • dudulin 3 A novel dudulin family member was identified using the TBLASTN algorithm ? as detailed in Example 4. This protein, herein termed dudulin 3, was found in both mouse and man, and had the closest homology to dudulin 2 of any protein or coding sequence identified. We therefore also termed dudulin 3 a 'dudulin 2 family member'.
  • the relationship of murine dudulin 2 to other murine and human dudulin family members is shown in Figure 5.
  • the position of the glutamine residue (Gin; Q) that is mutated in the dudulin 2 sequence of the present invention is conserved throughout the family of wild type dudulin proteins; this Gin being one of six conserved residues within a ten-residue sequence segment.
  • the amino-acid sequence for murine wild type dudulin 3 is shown in SEQ ID NO:32.
  • the amino-acid sequence for human wild type dudulin 3 is shown in SEQ ID NO:34.
  • the wild type residue of the modified human dudulin 2 protein is replaced by an amino acid with different size and/or polarity, i.e., a non-conservative amino acid substitution, as defined below.
  • Gin residue 395 of murine wild type dudulin 2 or Gin residue 395 of human wild type dudulin 2 is replaced by an amino acid residue other than Asn, and preferably is replaced by an amino acid residue selected from the group consisting of His, Arg, and Lys, and most preferably by Lys.
  • the murine dudulin 2 mutein of the present invention has the amino acid sequence shown in SEQ ID NO:4.
  • the human dudulin 2 mutein of the present invention has the amino acid sequence shown in SEQ ID NO:6.
  • the wild type residue of the modified human dudulin 3 protein is replaced by an amino acid with different size and/or polarity, i.e., a non-conservative amino acid substitution, as defined below.
  • Gin residue 366 of murine wild type dudulin 3 or Gin residue 364 of human wild type dudulin 3 is replaced by an amino acid residue other than Asn, and preferably is replaced by an amino acid residue selected from the group consisting of His, Arg, and Lys, and most preferably by Lys.
  • the murine dudulin 3 mutein of the present invention has the amino acid sequence shown in SEQ ID NO:36.
  • the human dudulin 3 mutein of the present invention has the amino acid sequence shown in SEQ ID NO:38.
  • the present invention is not limited to the mutation of the residue at position 395 of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6 or at a corresponding position in other dudulin 2 proteins, nor is it limited to the mutation of the residue at position 366 of the amino acid sequence shown in SEQ ID NO:36 or at position 364 of the amino acid sequence shown in SEQ ID NO:38 or at a corresponding position in other dudulin 3 proteins (see Fig.5). Rather it encompasses additional modifications in the amino acid sequence of the dudulin 2 protein or in the amino acid sequence of the dudulin 3 protein. Such mutations include single or multiple further amino acid substitutions, deletions and insertions.
  • the dudulin 2 polypeptide of the invention has at least 75 %, preferably at least 80 %, more preferably at least 90 %, even more preferably 95% and most preferably at least 99 % sequence identity with the wild type dudulin 2 sequence: in another preferred embodiment the dudulin 3 polypeptide of the invention has at least 75 %, preferably at least 80 %, more preferably at least 90 %, even more preferably 95% and most preferably at least 99 % sequence identity with the wild type dudulin 3 sequence.
  • the dudulin 2 polypeptide of the invention is identical with the corresponding wild type sequence except for a replacement of the Gin residue at position 395 of the amino acid sequence of dudulin 2, as shown in SEQ ID NO:4 and SEQ ID NO:6, or at the corresponding position in other dudulin 2 sequences.
  • Preferred modifications of the amino acid sequence in addition to the replacement at position 395 of the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:6, or at a corresponding position in other dudulins are at positions which are not conserved among the vertebrate dudulins (see Fig. 5). Conservative substitutions are defined below.
  • the dudulin 3 polypeptide of the invention is identical with the corresponding wild type sequence except for a replacement of the Gin residue at position 366 of the amino acid sequence of murine dudulin 3, as shown in SEQ ID NO:36, or at position 364 of the amino acid sequence of human dudulin 3 as shown in and SEQ ID NO:38, or at the corresponding position in other dudulin 2 sequences.
  • Preferred modifications of the amino acid sequence, in addition to the replacement at position 366 or 364 of sequences SEQ ID NO:36 and SEQ ID NO:38, respectively, or at a corresponding position in other dudulins are at positions which are not conserved among the vertebrate dudulins (see Fig. 5). Conservative substitutions are defined below.
  • an “isolated” or “purified” polypeptide or protein or biologically-active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the polypeptide or protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of dudulin 2 protein, and also preparations of dudulin 3 protein, in which the respective protein is separated from cellular components of the cells from which the dudulin 2 protein or the dudulin 3 protein is isolated or in which it is recombinantly produced.
  • a "mature" form of a polypeptide or protein may arise from a step of post-translational modification.
  • additional processes include, by way of non-limiting example, glycosylation, myristoylation, phosphorylation or proteolytic cleavage.
  • a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second comparison amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity").
  • the nucleic acid sequence homology may be determined as the degree of identity between two sequences.
  • the homology may be determined using computer programs known in the art, such as GAP software provided in the GCG program package. See, Needleman and Wunsch, 1970, J Mol Biol, 48: 443-453.
  • the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.
  • sequence identity refers to the degree to which two polynucleotide or polypeptide sequences are identical on a residue-by-residue basis over a particular region of comparison.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • Non-conservative substitutions are defined as exchanges of an amino acid by another amino acid listed in a different group of the five standard amino acid groups shown below:
  • Conservative substitutions are defined as exchanges of an amino acid by another amino acid listed within the same group of the five standard amino acid groups shown above. Three residues are parenthesized because of their special role in protein architecture. Gly is the only residue without a sidechain and therefore imparts flexibility to the chain. Pro has an unusual geometry which tightly constrains the chain. Cys can participate in disulfide bonds.
  • glutamine residue 395 of SEQ ID ⁇ O:2 or SEQ ID NO:8 is replaced by an amino acid of different size and/or polarity
  • lysine residue 395 of SEQ ID NO:4 or SEQ ID NO:6 is replaced by an amino acid of different size and/or polarity (excluding the wild type residue at this position)
  • this replacement is termed a non- conservative amino acid substitution.
  • Preferred embodiments of the present invention include the wild type murine dudulin 2 having amino acid sequence SEQ ID NO:2; wild type murine and human dudulin 3 having the amino acid sequence SEQ ID NO:32 and SEQ ID NO:34, respectively; mutant murine and human dudulin 2 having non-conservative substitution of lysine for glutamine at position 395 (murine mutated dudulin 2, SEQ ID NO:4; human mutated dudulin 2, SEQ ID NO:6); and mutant murine and human dudulin 3 having non- conservative substitution of lysine for glutamine at position 366 (murine mutated dudulin 3, SEQ ID NO:36) or position 364 (human mutated dudulin 3, SEQ ID NO:38).
  • the dudulin 2 expressed in the animal model of the present invention has the amino acid sequence shown in SEQ ID NO:4.
  • a novel "chimeric protein” or “fusion protein” comprises a novel dudulin 2 polypeptide linked to a non-dudulin 2 polypeptide (i.e., a polypeptide that does not comprise dudulin 2 or a fragment thereof).
  • a "chimeric protein” or “fusion protein” comprising a novel dudulin 3 polypeptide linked to a non-dudulin 3 polypeptide (i.e., a polypeptide that does not comprise dudulin 3 or a fragment thereof), a particular embodiment being a "chimeric protein” or “fusion protein” comprising such a dudulin 3 polypeptide linked to a dudulin 2 polypeptide.
  • the fusion protein is a GST-dudulin 2 fusion protein in which the dudulin 2 sequences are fused to the C-terminus of the GST (glutathione S-transferase) sequences.
  • Such fusion proteins can facilitate the purification of recombinant dudulin 2 polypeptides.
  • a further embodiment is the comparable GST-dudulin 3 fusion protein.
  • the fusion protein is a dudulin 2-immunoglobulin fusion protein in which the dudulin 2 sequences are fused to sequences derived from a member of the immunoglobulin protein family, especially Fc region polypeptides. Also contemplated are fusions of dudulin 2 sequences (mutant or wild type or functional fragments) fused to amino acid sequences that are commonly used to facilitate purification or labeling, e.g., polyhistidine tails (especially hexahistidine segments), FLAG tags, streptavidin.
  • the comparable dudulin 3 -immunoglobulin fusion proteins are provided by additional embodiments of the invention.
  • amino acid sequences of the present invention may be made by using peptide synthesis teclmiques well known in the art, such as solid phase peptide synthesis (see, for example, Fields et al., "Principles and Practice of Solid Phase Synthesis" in
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin (Ig) molecules i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F(ab') 2 fragments, and a Fab expression library.
  • antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
  • the light chain may be a kappa chain or a lambda chain.
  • Reference herein to antibodies includes a reference to all such classes, subclasses and isotypes of human antibody species.
  • An isolated protein of the invention intended to serve as an antigen, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that immunospecifically bind the antigen, using standard techniques for polyclonal and monoclonal antibody preparation.
  • the full-length protein can be used.
  • antigenic peptide fragments of the full-length protein can be used as immunogens.
  • Such fragments comprise, e.g. the modified amino acid(s) of the amino acid sequence of a mutein of the present invention, e.g. of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:36, or SEQ ID NO:38.
  • fragments comprise fragments of the wild type amino acid sequences as contemplated herein, e.g. the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:32, or SEQ ID NO:34.
  • the antigenic peptide comprises at least 7 amino acid residues, at least 10 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 30 amino acid residues of the afore-mentioned sequences.
  • Preferred epitopes encompassed by the antigenic peptide are regions of the protein that are located on its surface; commonly these are hydrophilic regions.
  • At least one epitope encompassed by the antigenic peptide is a region of the dudulin 2 or dudulin 3 polypeptide that is located on the surface of the protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis of a dudulin 2 or dudulin 3 polypeptide will indicate which regions of a dudulin 2 or dudulin
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. (See, for example, Hopp and Woods (1981) Proc Nat Acad Sci USA
  • Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologues thereof, are also provided herein.
  • a protein of the invention, or a derivative, fragment, analog, homologue or orthologue thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • polyclonal antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by one or more injections with the protein of the invention, a synthetic variant thereof, or a derivative of the foregoing.
  • An appropriate immunogenic preparation can contain, for example, the naturally occurring immunogenic protein, a chemically synthesized polypeptide representing the immunogenic protein, or a recombinantly expressed immunogenic protein.
  • the protein may be conjugated to a second protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin and soybean trypsin inhibitor.
  • the preparation can further include an adjuvant.
  • adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in humans such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory agents.
  • Additional examples of adjuvants which can be employed include MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the polyclonal antibody molecules directed against the immunogenic protein can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by Wilkinson (Wilkinson (2000) The Scientist, 14, 25-28).
  • MAb monoclonal antibody
  • CDRs complementarity determining regions
  • Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, 1975, Nature, 256:495.
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes can be stimulated to produce antibodies in vitro.
  • the immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice (Academic Press / Elsevier Science Sidcup, Kent, UK, 1986) at pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor (1984) J Immunol, 133, 3001; Brön et al., Monoclonal Antibody Production Techniques and Applications (Marcel Dekker, Inc., New York NY, USA, 1987) at pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard (1980) Anal. Biochem., 107:220.
  • antibodies having a high degree of specificity and a high binding affinity for the target antigen are isolated.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison (1994) Nature 368, 812-13) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies directed against the protein antigens of the invention can further comprise humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against an administered non-human immunoglobulin.
  • Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin.
  • Humanization can be performed following the method of Winter and co-workers (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. See, also, U.S. Patent No. 5,225,539, incorporated herein by reference. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al. (1986) Nature 321, 522-525; Riechmann et al. (1988) Nature 332, 323-327; and Presta (1992) Curr Op Struct Biol 2, 593-596).
  • Fc immunoglobulin constant region
  • Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B-cell hybridoma technique (see, Kozbor et al., 1983, Immunol Today, 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see, Cole et al., in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., 1985, pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter (1992) J Mol Biol 227, 381- 388; Marks et al. (1991) J Mol Biol 222, 581-597).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patents No.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen (see PCT publication WO94/02602).
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • nonhuman animal is a mouse, and is termed the Xenomouse ⁇ as disclosed in PCT publications WO-A-96/33735 and WO-A-96/34096.
  • This animal produces B cells which secrete fully human immunoglobulins.
  • the antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies.
  • the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv molecules.
  • a method of producing a nonhuman host exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598.
  • Such a host can be obtained by a method including deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
  • a method for producing an antibody of interest is disclosed in U.S. Patent No. 5,916,771.
  • the method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
  • the hybrid cell expresses an antibody containing the heavy chain and the light chain.
  • F fl h Fragments and Single Chain Antibodies According to the invention, techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see for example U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of F ab expression libraries (see for example Huse et al. (1989) Science 246, 1275-1281) to allow rapid and effective identification of monoclonal F ab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologues thereof. Antibody fragments that contain the idiotypes to a protein antigen may be produced by techniques known in the art including, but not limited to: (i) an F( ab' ) 2 fragment produced by pepsin digestion of an antibody molecule;
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for an antigenic protein of the invention.
  • the second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello (1983) Nature 305, 537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO-A-93/08829 and in Traunecker et al. (1991) EMBO J 10, 3655-3659.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy chain constant region (CHI) containing the site necessary for light-chain binding present in at least one of the fusions.
  • CHI first heavy chain constant region
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 region of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab') 2 bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al. ((1985) Science 229, 81) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • Fab' fragments can be recovered directly from E. coli and chemically coupled to form bispecific antibodies.
  • Shalaby et al. ((1992) J Exp Med 175, 217-225) describe the production of a fully humanized bispecific antibody F(ab') 2 molecule.
  • Each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.
  • the bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V H and V L domains of one fragment are forced to pair with the complementary V and V H domains of another fragment, thereby forming two antigen-binding sites.
  • V H and V L domains of one fragment are forced to pair with the complementary V and V H domains of another fragment, thereby forming two antigen-binding sites.
  • sFv single-chain Fv
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared (Tutt et al. (1991) J Immunol 147, 60-69).
  • bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention.
  • an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fc RI (CD64), Fc RII (CD32) and Fc RIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen.
  • Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen.
  • antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • a cytotoxic agent or a radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.
  • Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).
  • Heteroconjugate antibodies are also within the scope of the present invention.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO-A-91/00360; WO-A-92/20373; EP-A3-0308936).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.
  • the antibody of the invention can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody.
  • cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
  • the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al. (1992) J Exp Med 176; 1191-1195 and Shopes (1992) J Immunol 148, 2918-2922).
  • ADCC antibody-dependent cellular cytotoxicity
  • Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al.
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities (see Stevenson et al. (1989) Anti-Cancer Drug Design 3, 219-230).
  • the invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (e.g., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • a variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212 Bi, 131 1, 131 In, 90 Y, and 186 Re.
  • Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds
  • SPDP N-succinimidyl-3-(2-pyridyldithiol) propionate
  • IT imino
  • a ricin immunotoxin can be prepared as described in Vitetta et al. (1987) Science 238, 1098-1104. Carbon-14- labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-
  • DTPA is an exemplary chelating agent for conjugation of radionucleotide to the antibody (see WO-A-94/11026).
  • the antibody in another embodiment, can be conjugated to a "receptor" (such streptavidin) for utilization in tumor pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is in turn conjugated to a cytotoxic agent.
  • a "receptor” such streptavidin
  • a "ligand” e.g., avidin
  • vectors and Cells Expressing Dudulin 2 Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a dudulin 2 protein, or derivatives, fragments, analogs or homologues thereof.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vectors refers to a circular double stranded circular DNA molecule into which additional DNA segments can be ligated.
  • a viral vector Another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as "expression vectors”.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) dudulin 2 protein and/or dudulin 3 protein.
  • the invention further provides methods for producing dudulin 2 protein using host cells of the invention, and methods for producing dudulin 3 protein using host cells of the invention.
  • the method comprises culturing host cells of the invention (into which a recombinant expression vector encoding dudulin 2 protein has been introduced) in a suitable medium such that dudulin 2 protein is produced: in another embodiment, the method further comprises isolating dudulin 2 protein from the medium or the host cell.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding dudulin 3 protein has been introduced) in a suitable medium such that dudulin 3 protein is produced: in yet another embodiment, the method further comprises isolating dudulin 3 protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which dudulin 2 protein-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous dudulin 2 sequences have been introduced into their genome or homologous recombinant animals in which endogenous dudulin 2 sequences have been altered.
  • Such animals are useful for studying the function and/or activity of dudulin 2 protein and for identifying and/or evaluating modulators of dudulin 2 protein activity.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which dudulin 3 protein-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous dudulin 3 sequences have been introduced into their genome or homologous recombinant animals in which endogenous dudulin 3 sequences have been altered.
  • Such animals are useful for studying the function and/or activity of dudulin 3 protein and for identifying and/or evaluating modulators of dudulin 3 protein activity.
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • transgenic animals include non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, chickens, amphibians, etc. Standard methods are known in the art that may be used in conjunction with the polynucleotides and of the invention and methods described herein to produce a transgenic animal expressing a modified dudulin 2 of the invention, and/or a dudulin 3 of the invention.
  • the present invention provides a non-human animal model which expresses a dudulin 2 protein modified as compared to the amino acid sequence of the wild type protein.
  • the dudulin 2 expressed may have similarity in sequence and secondary structure to a vertebrate dudulin 2, including, but not limited to mammalian dudulin 2 proteins, preferably of bovine, equine, porcine, ovine, canine, feline, or rodent (particularly rat, and preferably mouse) origin.
  • the animal is preferably from a genus selected from the group consisting of Mus (e.g., mice), Rattus (e.g., rats), Oryctologus (e.g., rabbits) and Mesocricetus (e.g., hamsters).
  • the animal is a mouse.
  • Animals carrying a mutated dudulin 2 gene expressing said modified dudulin 2 exhibit one or more of the following phenotypic features:
  • the phenotype is a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal in conjunction with an elevated red blood cell distribution width or an elevated reticulocyte-count.
  • Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) of affected animals are significantly below average, whereas red blood cell distribution width (RDW) is significantly above average. The total count of erythrocytes is above normal.
  • phenotype refers to a collection of morphological, physiological, behavioral and biochemical traits possessed by a cell or organism that results from the interaction of the genotype and the environment.
  • the animal model of the present invention displays readily observable abnormalities, preferably exhibiting at least one and preferably all of the above-listed phenotypic features or traits.
  • mice were generated carrying a point mutation in the coding region of the mouse dudulin 2 gene as shown in SEQ ID NO:3, thereby replacing the Gin residue at position 395 in the extracellular domain of the protein with Lys.
  • Amino acid position 395 according to the present invention refers to the non- mature dudulin 2 protein, as numbered in SEQ ID NO:4.
  • the mature protein is encompassed by the present invention and may be expressed in the animal model of the present invention. This modification of dudulin 2 results in the above mentioned phenotypic features.
  • the residue at position 395 in the amino acid sequence of SEQ ID NO:4 and SEQ ID NO:6 is conserved between mice and humans, as can be seen in the alignment of the mouse and human sequences shown in Figure 4.
  • the residue 395 in the amino acid sequence of SEQ ID NO:4 is not only conserved in dudulin 2 proteins but also in all currently known family members of the dudulin 2 family (Fig. 5), including dudulin 3, identified herein as a dudulin 2 family member.
  • the non-human animal model of the present invention carries a nucleic acid sequence encoding dudulin 2, whereby the codon for the amino acid at position 395 of the amino acid sequence shown in SEQ ID NO:4 and SEQ ID NO:6 or the codon corresponding to said position in other dudulin 2 proteins, which encodes a glutamine (Gin) in the wild type, is mutated to encode a different amino acid.
  • the animal model of the present invention expresses the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:6.
  • the animal model of the invention carries a modified dudulin 2 nucleic acid sequence derived from a vertebrate, preferably from a mammal, in particular from mouse.
  • the nucleic acid sequence is derived from the nucleic acid sequence shown in the sequence listing as SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:5.
  • the animal model of the invention preferably expresses a dudulin 2 mRNA (e.g., as shown in SEQ ID NO:4) in the following tissues: brain, olfactory lobes, cerebrum, cerebellum, pituitary gland, medulla oblongata, medulla spinalis, eye, nose epithelium, trachea, lung, tongue, esophagus, salivary gland, stomach, pancreas, small intestine, large intestine, appendix, rectum, thymus, heart, mesenterium, liver, gall bladder, spleen, kidney adrenal gland, bladder, uterus, ovary, testis, epididymis, prostate, sternum, bone marrow, skin, adipose tissue and skeletal muscle.
  • a dudulin 2 mRNA e.g., as shown in SEQ ID NO:4
  • tissues e.g., as shown in SEQ ID NO:4
  • dudulin 2 expression was detected in lymph nodes and whole blood. In all tissues in which dudulin 2 expression occurs in the animal model of the invention, it is preferable if the dudulin 2 expressed is the modified protein of the invention. However, animals which express the modified dudulin 2 protein in some, but not all cells, which are termed cellular mosaic animals, are also contemplated.
  • the present invention further provides for inbred successive lines of animals carrying the mutant dudulin 2 nucleic acid of the present invention that offer the advantage of providing a virtually homogenous genetic background.
  • a genetically homogenous line of animals provides a functionally reproducible model system for disorders or symptoms associated with dudulin 2 activity deficiency, preferably relating to hematological disorders, and iron uptake capacity.
  • the animal models of the present invention may use any of the mutein dudulins described above, and is not limited to the modification of the residue at position 395 of the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:6, respectively, or at a corresponding position in other dudulin 2 homologues and orthologues, so long as the mutein used imparts the desired hematologic-deficient phenotype to the animal of the present invention. Rather it encompasses additional dudulin mutations, preferably mutations in dudulin 2, as long as they do not result in a loss of the phenotype. Such mutations include single or multiple further amino acid substitutions, deletions and insertions.
  • the animal models of the present invention may use the mutein dudulin 3 proteins described above, bearing a modification of the residue at position 366 of the amino acid sequence shown in SEQ ID NO:32 or position 364 of the amino acid sequence shown in SEQ ID NO:34, such as SEQ ID NO:36 and SEQ ID NO: 38, respectively.
  • Amino acid insertional derivatives of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product.
  • Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • the dudulin 2 expressed in the animal model invention has a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or most preferably 99%, with the wild type dudulin 2 sequence from vertebrate, preferably from mammals, more preferably from bovine, and even more preferably from rat and most preferably from mouse (SEQ ID NO:2).
  • the dudulin 2 expressed in the animal model of the invention is human dudulin 2 (SEQ ID NO: 6 or SEQ ID NO:8) or has a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or most preferably 99%, with the human dudulin 2 sequences in SEQ ID NO:6 or SEQ ID NO:8, excluding the wild type human dudulin 2 sequence.
  • the animal model expresses a polypeptide as shown in SEQ ID NO:4 or SEQ ID NO:6.
  • Preferred modifications of the dudulin 2 amino acid sequence in the animal models of the present invention, in addition to the modification at position 395 of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO: 6, are at positions which are not conserved among the vertebrate dudulin 2 proteins, as described above.
  • the animal model of the invention may carry a mutated dudulin 2 nucleic acid according to the present invention derived from the same species or from a different species.
  • the mutated dudulin 2 nucleic acid of the present invention is homozygous in the animals of the present invention.
  • transcription of the mutated dudulin 2 gene of the present invention is under the control of the promoter sequence controlling transcription of the endogenous wild type dudulin 2 sequence of the animal, although a different promoter may be used.
  • the animals of the invention can be produced by using any technique known to the person skilled in the art; including but not limited to micro-injection, electroporation, cell gun, cell fusion, micro-injection into embryos of teratocarcinoma stem cells or functionally equivalent embryonic stem cells.
  • the animals of the present invention may be produced by the application of procedures, which result in an animal with a genome that incorporates/integrates exogenous genetic material in such a manner as to modify or disrupt the function of the normal dudulin 2 gene or protein.
  • the preferred procedure for generating animal models of this invention is according to Example 1.
  • the procedure may involve obtaining genetic material, or a portion thereof, which encodes a dudulin 2.
  • the isolated native sequence is then genetically manipulated by the insertion of a mutation appropriate to replace the residue at position 395 of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:8.
  • the manipulated construct may then be inserted into embryonic stem cells, e.g., by electroporation.
  • the cells subjected to said procedure are screened to find positive cells, i.e., cells that have integrated into their genome the desired construct encoding an altered dudulin 2.
  • the positive cells may be isolated, cloned (or expanded) and injected into blastocysts obtained from a host animal of the same species or a different species.
  • blastocysts positive cells are injected into blastocysts from mice, the blastocysts are then transferred into a female host animal and allowed to grow to term, following which the offspring of the female are tested to determine which animals are transgenic, i.e., which animals have an inserted exogenous mutated DNA sequence.
  • One method involves the introduction of the recombinant gene at the fertilized oocyte stage ensuring that the gene sequence will be present in all of the germ cells and somatic cells of the "founder" animal.
  • the term "founder animal” as used herein means the animal into which the recombinant gene was introduced at the one cell embryo stage.
  • the animals of the invention can also be used as a source of primary cells from a variety of tissues, for cell culture experiment, including but not restricted to, the production of immortalized cell lines by any methods known in the art, such as retroviral transformation.
  • Cells from the animals may advantageously exhibit desirable properties of both normal and transformed cultured blood cells, i.e., they will be normal or nearly normal morphologically and physiologically, but can be cultured for long, and perhaps indefinite periods of time.
  • the present invention provides such primary cells and cell lines derived therefrom, obtained from the animals of the present invention. These primary cells or cell lines derived thereof may be used for the construction of an animal model of the present invention.
  • cell lines may be prepared by the insertion of a nucleic acid construct comprising the nucleic acid sequence of the invention or a fragment thereof comprising the codon imparting the above described phenotype to the animal model of the invention (vide infra).
  • Suitable cells for the insertion include primary cells harvested from an animal as well as cells, which are members of an immortalized cell line.
  • Recombinant nucleic acid constructs of the invention, described below, may be introduced into the cells by any method known in the art, including but not limited to, transfection, retroviral infection, micro-injection, electroporation, transduction or DEAE-dextran.
  • Cells, which express the recombinant construct may be identified by, for example, using a second recombinant nucleic acid construct comprising a reporter gene, which is used to produce selective expression.
  • Cells that express the nucleic acid sequence of the invention or a fragment thereof may be identified indirectly by the detection of reporter gene expression.
  • compositions The mutated nucleic acid sequences and muteins of this invention, and antibodies thereto, may be used in pharmaceutical compositions, when combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed.), Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, PA 1990), a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), fransmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ, U.S.A.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • Suitable binders include starch, magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • diluents include, without limitation, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • the compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixers, tinctures, suspensions, syrups and emulsions.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • Injectable compositions are preferably aqueous isotonic solutions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Additionally, one approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.
  • preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines.
  • a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.
  • Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • the dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed.
  • An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Oral dosages of the present invention when used for the indicated effects, will range between about 0.05 to 1000 mg/day orally.
  • the compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient.
  • Effective plasma levels of the compounds of the present invention range from 0.002 mg to 50 mg per kg of body weight per day.
  • Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • compositions may contain 0.1-99%, preferably 1-70% of the dudulin 2 or 3 polypeptide.
  • compositions can be provided with an adjuvant.
  • adjuvants are discussed above.
  • adjuvants can be used to increase the immunological response, depending on the host species, include Freund's
  • mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • animals are injected with antigen using several injections in a series, preferably including at least three booster injections.
  • the animals of the present invention present a phenotype whose characteristics are representative of many symptoms associated with dudulin 2 deficiency associated disorders, therefore making the animal model of the present invention a particularly suitable model for the study of these diseases.
  • the phenotype of dudulin 2 deficient mice is a microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal in conjunction with an elevated red blood cell distribution width or an elevated reticulocyte-count. This phenotype may arise from different pathophysiological processes.
  • the phenotype is not an anemia in a strict sense, as there is no reduced hemoglobin level.
  • the association of microcytosis with an elevated RBC distribution width or an elevated reticulocyte-count is a common finding in compensated anemias due to bleeding disorders or iron deficiency.
  • Anemias are caused by disturbances of the hematopoietic system or are secondary to an underlying systemic disease. To dissect these possibilities it is important to understand characteristics of the chronic diseaserelated anemia. Chronic anemias show low serum iron, low iron binding capacity, slightly reduced RBC lifespan, reduced transferrin saturation and normal tissue iron storage. The erythropoietin level is inversely correlated with the degree of anemia. The response to erythropoietin is blunted if the hematopoietic system is disturbed. Alternatively, the response might also be blunted if there is a systemic disorder. In this case elevated levels of IL1, TNF and other inflammatory cytokines cause suppression of the hematopoietic system.
  • erythropoietin Inadequately low levels of erythropoietin are due to renal tissue damage, whereas elevated levels are due to failure of metabolizing the hormone by the hematopoietic cells and thus indicative of an insufficiency of the hematopoietic system.
  • the list of systemic disorders affecting the hematopoietic system is long and includes chronic renal failure, endocrine diseases such as Sheehan's syndrome or gonadal, adrenal, parathyroid and thyroid dysfunctions, gastrointestinal and hepatic disorders, arthritis, infection and cancer.
  • the animal model of the invention may be used to study such diseases, their symptoms, and current and experimental methods of diagnosis and treatments.
  • the animal model of the invention presents a phenotype characterized by red blood cells with mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) significantly below average.
  • MCV mean corpuscular volume
  • MCH mean corpuscular hemoglobin
  • Red blood cell distribution width (RDW) and reticulocyte-counts are significantly above average.
  • Red blood cell (RBC) counts are slightly but reproducibly above average (see Example 2).
  • the animals of the present invention can also be used to identify early diagnostic markers for diseases associated with dudulin 2 deficiency.
  • Surrogate markers including but not limited to ribonucleic acids or proteins, can be identified by performing procedures of proteomics or gene expression analysis known in the art.
  • procedures of proteomics analysis include, but are not restricted to, ELISA, 2D-gel, protein microarrays or mass spectrophotometric analysis of any organ or tissue samples, such as blood samples, or derivatives thereof, preferably plasma, at different age or stage of dudulin 2 activity deficiency associated disease development, or symptom thereof.
  • gene expression analysis procedures include, but are not restricted to, differential display, cDNA microarrays, analysis of quality and quantity of ribonucleic acids species from any organ or tissue samples, such as blood samples, or derivatives thereof, at different age or stage of development of dudulin 2 activity deficiency associated disease, or symptom thereof.
  • the animal model of the present invention can be used to monitor the activity of agents useful in the prevention or treatment of the above-mentioned hematological diseases.
  • the agent to be tested can be administered to an animal of the present invention and various phenotypic parameters can be measured or monitored.
  • the animals of the invention may be used to test therapeutics against any disorders or symptoms that have been shown to be associated with dudulin 2 deficiency.
  • the animals of the present invention can also be used as test model systems for materials, including but not restricted to chemicals and peptides, particularly medical drugs, suspected of promoting or aggravating the above-described diseases associated with dudulin 2 deficiency.
  • the material can be tested by exposing the animal of the present invention to different time, doses and/or combinations of such materials and by monitoring the effects on the phenotype of the animal of the present invention, including but not restricted to growth rates, dudulin 2 levels, iron uptake, red blood cell production, as well as information processing capacities and cognitive functions.
  • the animals of the present invention may be used for the dissection of the molecular mechanisms of the dudulin 2 pathway, that is for the identification of downstream genes or proteins thereof regulated by dudulin 2 activity and deregulated in dudulin 2 activity deficiency associated disorders.
  • this can be done by performing differential proteomics analysis, using techniques including but not restricted to 2D gel analysis, protein chip microarrays or mass spectrophotometry, on tissues of the animal of the present invention which express dudulin 2 and which respond to dudulin 2 stimuli.
  • the animals of the present invention can also be used as test model systems for materials, including but not restricted to chemicals and peptides, particularly medical drugs, suspected of promoting or aggravating the above-described diseases associated with dudulin 2 or dudulin 3 deficiency.
  • the material can be tested by exposing the animal of the present invention to different time, doses and/or combinations of such materials and by monitoring the effects on the phenotype of the animal of the present invention, including but not restricted to microcytosis, a mean corpuscular volume below normal, a mean corpuscular hemoglobin below normal, an elevated reticulocyte-count and/or elevated red blood cell distribution.
  • the animals of the present invention may be used for the dissection of the molecular mechanisms of the dudulin 2 or dudulin 3 pathway, that is for the identification of downstream genes or proteins thereof regulated by dudulin 2 or dudulin 3 activity and deregulated in the dudulin 2 or dudulin 3 activity deficiency associated disorders.
  • this can be done by performing differential proteomics analysis, using techniques including but not restricted to 2D gel analysis, protein chip microarrays or mass spectrophotometry, on tissues of the animal of the present invention which express dudulin 2 or dudulin 3 and which respond to the dudulin 2 or dudulin 3 stimuli.
  • the animal model of the present invention can be used to identify and clone so-called modifier genes, which are able to modify, aggravate, reduce or inhibit the phenotype associated with a dudulin 2 activity deficiency.
  • the animal model of the present invention can be mated to mice of different strains carrying a different genetic background, which gives the possibility to map the genes modifying the phenotype.
  • the animal model of the present invention when produced in an C3H/HeJ inbred strain background can be bred to C57/BL6J inbred mice. The hybrid animals of this progeny, are then further bred, either in back-cross strategy with C57/BL6J inbred mice again, or in an intercross strategy between each other.
  • the modifier gene can then be mapped and cloned by using microsattelites or a single nucleotide polymorphism (SNP) strategy on the mice resulting from the backcross or intercross breeding that have been grouped with respect to their phenotype intensity.
  • SNP single nucleotide polymorphism
  • An exemplary method for detecting the presence or absence of dudulin 2 in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting dudulin 2 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes dudulin 2 protein such that the presence of dudulin 2 is detected in the biological sample.
  • a compound or an agent capable of detecting dudulin 2 protein or nucleic acid e.g., mRNA, genomic DNA
  • An agent for detecting dudulin 2 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to dudulin 2 mRNA or genomic DNA.
  • An exemplary method for detecting the presence or absence of dudulin 3 in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting dudulin 3 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes dudulin 3 protein such that the presence of dudulin 3 is detected in the biological sample.
  • a compound or an agent capable of detecting dudulin 3 protein or nucleic acid e.g., mRNA, genomic DNA
  • An agent for detecting dudulin 3 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to dudulin 3 mRNA or genomic DNA.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having, or at risk of developing a disease or disorder associated with aberrant dudulin expression or activity, particularly aberrant dudulin 2 expression or activity and/or aberrant dudulin 3 expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having, or at risk of developing a disorder associated with dudulin protein, nucleic acid expression or activity, wherein the dudulin with which the dudulin is associated is dudulin 2 and/or dudulin 3.
  • the prognostic assays can be utilized to identify a subject having, or at risk for developing a hematological disease or disorder, particularly one presenting or characterized by one or more of the following medical conditions: mean corpuscular volume (MCV) below normal, mean corpuscular hemoglobin (MCH) below normal, or red blood cell distribution width (RDW) above normal.
  • MCV mean corpuscular volume
  • MH mean corpuscular hemoglobin
  • RW red blood cell distribution width
  • the invention provides a method for identifying a disease or disorder associated with aberrant dudulin 2 expression or activity in which a test sample is obtained from a subject and dudulin 2 protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of dudulin 2 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant dudulin 2 expression or activity.
  • dudulin 2 protein or nucleic acid e.g., mRNA, genomic DNA
  • the present invention provides a method for identifying a disease or disorder associated with aberrant dudulin 3 expression or activity in which a test sample is obtained from a subject and dudulin 3 protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of dudulin 3 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant dudulin 3 expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., blood, plasma, serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant dudulin 2 expression or activity, and/or to treat a disease or disorder associated with aberrant dudulin 3 expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.
  • Agents, or modulators that have a stimulatory or inhibitory effect on dudulin 2 activity can be administered to individuals to treat (prophylactically or therapeutically) dudulin 2- mediated disorders.
  • Agents, or modulators that have a stimulatory or inhibitory effect on dudulin 3 activity e.g., dudulin 3 gene expression
  • a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) dudulin 3 -mediated disorders.
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of dudulin 2 protein, expression of dudulin 2 nucleic acid, or mutation content of dudulin 2 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. Similarly, the activity of dudulin 3 protein, expression of dudulin 3 nucleic acid, or mutation content of dudulin 3 genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • the present invention also provides a diagnostic method for dudulin 2 activity deficiency and a diagnostic method for dudulin 3 activity deficiency.
  • Patients' peptide material particularly that in or from blood, serum or plasma, is subjected to analysis for one or more of the amino acid sequences of the present invention.
  • the peptide material may be analyzed directly or after extraction, isolation and/or purification by standard methods.
  • the diagnostic method comprises the identification of the modified dudulin 2, whereby the modification is associated with the replacement of an amino acid at a position corresponding to position 395 in the amino acid sequence shown in SEQ ID NO:8.
  • diagnostic methods include those employing detection of the modified dudulin 2 by its failure to activate a biological pathway.
  • the diagnostic methods of the invention also include those employing detection of the modified dudulin 2 by its activity in competing with and blocking the action of native dudulin 2.
  • Methods of identifying the modified dudulin 2 include any methods known in the art which are able to identify altered conformational properties of the amino acid sequence of the present invention compared to those of the wild type dudulin 2.
  • the method exploits the failure of another protein to recognize the modified protein, examples being antibodies directed to an epitope of wild type dudulin 2 that incorporates residue 395 of SEQ ID NO:2 or SEQ ID NO:8, and dudulin 2 receptors in which this portion of the molecular surface of wild type dudulin 2 is recognized or involved in dudulin 2 activation.
  • the principle of the diagnostic method is the detection of a nucleic acid sequence encoding the modified dudulin 2 of the invention. This can be accomplished by simple sequencing of PCR-products generated with primers dudulin-9 and dudulin-10 (SEQ ID NO:25 and SEQ ID NO:26). Other techniques include, but are not restricted to any methods known in the art using nucleic acid hybridizing properties, such as Northern blot, Southern blot, nucleic acid (genomic DNA, cDNA, mRNA, synthetic oligonucleotides) standard methods employing microarrays, and patterns of nucleic acid digestion by known restriction enzymes, preferably Bsgl or BspMl.
  • the diagnostic method comprises the identification of the modified dudulin 3, whereby the modification is associated with the replacement of an amino acid at a position corresponding to position 364 in the amino acid sequence shown in SEQ ID NO:34.
  • diagnostic methods include those employing detection of the modified dudulin 3 by its failure to activate a biological pathway.
  • the diagnostic methods of the invention also include those employing detection of the modified dudulin 3 by its activity in competing with and blocking the action of native dudulin 3.
  • Methods of identifying the modified dudulin 3 include any methods known in the art which are able to identify altered conformational properties of the amino acid sequence of the present invention compared to those of the wild type dudulin 3.
  • the method exploits the failure of another protein to recognize the modified protein, examples being antibodies directed to an epitope of wild type dudulin 3 that incorporates residue 366 of SEQ ID NO:32 or residue 364 of SEQ ID NO:34, and dudulin 3 receptors in which this portion of the molecular surface of wild type dudulin 3 is recognized or involved in dudulin 3 activation.
  • the principle of the diagnostic method is the detection of a nucleic acid sequence encoding the modified dudulin 3 of the invention.
  • Other teclmiques include, but are not restricted to any methods known in the art using nucleic acid hybridizing properties, such as Northern blot, Southern blot, nucleic acid (genomic DNA, cDNA, mRNA, synthetic oligonucleotides) standard methods employing microarrays, and patterns of nucleic acid digestion by known restriction enzymes, preferably Bsgl or BspMl.
  • mice mutants To produce mouse mutants, a C3H/HeJ male mouse (The Jackson Laboratory, Bar Harbor ME, U.S.A.) was injected intraperitoneally three times in weekly intervals between 8-10 weeks of age with the highly mutagenic agent, ethyl-nitroso-urea (ENU) (Serva Electrophoresis GmbH, Heidelberg, Germany) at 90mg/kg body weight. 50 days after the last injection the injected male mouse was regularly mated to wild type C3H/HeJ female partners. The FI progeny (up to 100 offspring) were then analyzed for phenotypes of dominant traits.
  • ENU ethyl-nitroso-urea
  • F3 Progeny -- Breeding Scheme We generated F3 progeny, using the breeding scheme shown in figure 1 A. All breeding partners were older than 8 weeks (56 days); preferably females were between 8-12 weeks old and males were between 8-16 weeks old.
  • Each ENU-male produced as described above was used to generate no more than 30 male and 30 female pups, which were interbred as described below.
  • Blood was sampled according to a standard operating protocol: Animals are not fed the day before blood is withdrawn. A heparinized capillary is inserted into the vessel network behind the glass body of the right eye with a twisting movement, passing via the nose side and by the glass body of the right eye. A predetermined blood volume is withdrawn. The blood volume is dependent on mouse line and age. Generally, for C3H/HeJ mice, we draw 600 ⁇ l of whole blood and for C57BL/6J mice we draw 500 ⁇ l whole blood. Blood is collected into heparin tubes and EDTA tubes. The heparin tube is inverted and analyzed when relevant (not necessary for this mouse phenotype).
  • the EDTA tubes were processed in the ABC Analyzer (described below).
  • MCH mean corpuscular hemoglobin (a measure of the hemoglobin content of red blood cells)
  • RW red blood cell distribution width (a measure of the variance in MCV values [cell sizes])
  • the F3 Founder male was identified by following values, all measured at 134 days. These values stood out in comparison with control mice, i.e., non-mutagenized male animals of the same strain, baseline values taken at 134 days (average of at least ten control animals [C3H/HeJ]).
  • the values obtained for the dudulin 2 founder animal placed the animal within the 99th percentile (MCH, MCV) or the 1st percentile (RDW) of animals tested for these parameters, a 'wild type limit value' being obtained for each parameter from the animals excluded on this percentile basis. Values obtained for dudulin 2 phenotype mice were at least two standard deviations (SD) lower (MCH, MCV) or higher (RDW) than the mean of corresponding measurements of 10 wild type control animals (C3H/HeJ).
  • a value for MCH (male) of 15.79 was the norm, with values more than two standard deviations from this value being considered abnormal (i.e. significantly higher or lower than the norm);
  • a value for MCN (male) of 50.15 was the norm, with values more than two standard deviations from this value being considered abnormal (i.e. significantly higher or lower than the norm);
  • a value for RDW (male) of 15.97 was the norm, with values more than two standard deviations from this value being considered abnormal (i.e. significantly higher or lower than the norm).
  • mice with the mutant dudulin 2 phenotype had MCH and MCN values that were lower than the baseline threshold value, and an RDW value higher than the baseline threshold value.
  • Reticulocyte-counts were determined as follows: blood samples were taken as previously described (Primary Screen). Reticulocytes were counted by flow cytometry using a fluorescence-labeled anti-CD71 antibody.
  • Figure 6 shows the percentage of reticulocytes in blood of dudulin 2 animals (C3H/HeJ-C57BL/6J RIC, see breeding scheme Fig. IB). Animals with a homozygous dudulin 2 mutation (mut) show an elevated average level of 5,2% reticulocytes compared to animals heterozygous (hz) or wild type (wt) for the dudulin 2 mutation, which both show average values around 3,4 %. The presented values are averages with standard deviations of four (4) animals for each category. Student t-test analysis indicated the reliability of the elevated reticulocyte values observed in homozygous mutant animals, compared with unaffected animals (wild type + heterozygous).
  • the F5 genomic DNA was purified from 1 cm long pieces of tails of F5 mice by using the "DNeasy 96 Tissue Kit” (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
  • a first PCR was run with 3 ⁇ l Template-DNA (10 pmol/ ⁇ l) and a mix of both primers (1 ⁇ l each) in 25 ⁇ l total volume.
  • the following template DNAs were used: • Pool-DNA of the respective two outcross-strains,
  • a Touch-Down PCR Program with 44 cycles was used to amplify DNA fragments. Received amplicons were visualized via an agarose gel electrophoresis, purified and resuspended in 50 ⁇ l water. 3 ⁇ l were used for a sequencing reaction with 1 ⁇ l of sequencing primer in a 10 ⁇ l volume.
  • FIG. 1 shows a plot of the scores for chromosome 1 obtained for the affected animals. The only chromosome that contained scores of 1 was chromosome 1.
  • the Xpg marker has the sequence shown in SEQ ID NO:l 1
  • the Gos2 marker has the sequence shown in SEQ ID NO: 12
  • Primer positions are underlined and the SNP-position is highlighted.
  • the DlMit433 marker is known and has the sequence:
  • the Dllng39 marker has the sequence:
  • Gcc (SEQ ID NO: 14).
  • the region proximal of DlMit433 and distal of Dllng39 is excluded from being a candidate region. Genes between these markers (also partially) can be responsible for the phenotype. However, by examination of the markers Ralb and Dllng57, we determined that the minimal genome area to be considered to find the mutation of interest fell between the Ralb and Dllng57 markers.
  • the sequence of the Ralb marker is:
  • Primer positions are underlined and the SNP-position is highlighted.
  • the sequence of the Dllng57 marker is:
  • the genome between the DlMit433 and Dllng39 was then searched for genes (by comparison with public mouse and human gene databases) that fell in this region. In this instance there were nine known genes recorded within this region. Among the genes contained in this region is the dudulin 2 gene.
  • a sequence for the mouse dudulin 2 gene was found in GenBank at Accession Number AY029586 (this sequence is incorrect and is shown as SEQ ID NO:9).
  • the correct mouse wild type cDNA sequence is shown in SEQ ID NO:l - this sequence is shared for C3H (e.g. CH3/HeJ) and AKR (e.g. AKR J) strains.
  • the sequence of the C57/BL6 (e.g. 'C57/BL6/J) mouse differs from that for CH3 and AKR in 17 nucleotide positions (see SEQ ID NO: 10, which is the cDNA encoding dudulin 2 in C57/BL6/J mice).
  • dudulin-1 5'-AGTCCCTGTGTTTGCAAGAG-3' (SEQ ID NO:17) dudulin-2 5'-TCCTGGAAAGTCACTTGAGC-3* (SEQ ID NO: 18) dudulin-3 5'-GTGGACAGTGATGGCAGTCT-3' (SEQ ID NO: 19) dudulin-4 5'-CCAACTCCCTGCTGTGTAAG-3' (SEQ ID NO:20) dudulin-5 5'-CAGCATCAGAGCCATCTTG-3' (SEQ ID NO:21) dudulin-6 5'-GCAGGTACACTAGGGACAGC-3' (SEQ ID NO:22) dudulin-7 5'-CATCCGAGACGTTCTACAGC-3' (SEQ ID NO:23) dudulin-8 5'-GTCTTCCTCCTCAGTGCTCA-3' (SEQ ID NO:24) dudulin-9 5'-TCTGGCAGAGCATGAACATA-3 ' (SEQ ID NO:25) dudulin-1
  • the underlined primers are the ones that flanked the mutation: i.e. primers dudulin-9 and dudulin-10, SEQ ID NO:25 and SEQ ID NO:26, respectively.
  • PCR amplicons were purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. PCR products were sequenced using forward/reverse PCR primers and "Big Dye” thermal cycle sequencing Kit (ABI PRISM, Applied Biosystems, Foster City, CA, U.S.A.). The reaction products were analyzed on an ABI 377 DNA sequencing device.
  • both copies of the dudulin 2 gene had the wild type sequence (SEQ ID NO:l) or the animals were heterozygotes with one wild type copy of the dudulin 2 gene (as shown in SEQ ID NO:l) and one mutated copy (as shown in SEQ ID NO: 3). Because this is a recessive trait, physiological effects of the mutation are not apparent in heterozygotes.
  • the SNP at position 1228 was a coding SNP and caused an amino acid change from Gin to Lys.
  • dudulin 2 sequences presented below were deduced from public domain data and partial sequencing of the dudulin genomic region: the dudulin 2 exons were individually identified, amplified and sequenced: CACGCGTCCGGGCTCCTCCAAGCGGCCGGCTGCCGAGGCACTGCTATGTCGGGGGAGATGGACAAGCCGC TGATCAGCCGCCGCCTAGTGGACAGTGATGGCAGTCTGGCTGAGGTCCCCAAGGAGGCCCCCAAAGTGGG CATCCTGGGCAGTGGGGATTTTGCCCGTTCCCTGGCCACACGCCTGGTGGGCTCTGGCTTCAGTGTGGTG GTGGGGAGCCGTAACCCCAAACGCACGGCTGGCCTCTTCCCCTCCTTAGCTCAAGTGACTTTCCAGGAGG AAGCCGTGAGCTCTCCAGAGGTCATCTTTGT
  • dudulin 2 mouse protein for C3H/HeJ or AKR J mouse strains.
  • An embodiment of the present invention contains a mutated dudulin 2 gene; for example, wherein the coding sequence is that of C3H/HeJ or AKR/J mouse strains, wherein C 1228 is altered to A 1228, causing a codon change (CAG to AAG):
  • a cDNA sequence containing the complete coding sequence for the C57/BL6/J mouse wild type dudulin 2 gene is shown below with the location for a SNP corresponding to the mutant disclosed herein indicated as underlined.
  • dudulin 2 sequences presented below were deduced from public domain data and by partial sequencing of the dudulin genomic region from mouse strain C57/BL6/J: the dudulin2 exons were individually identified, amplified and sequenced:
  • An embodiment of the present invention is a polynucleotide comprising the complete coding sequence for the C57/BL6/J mouse mutant dudulin 2 gene (as shown below with the location of the mutant SNP disclosed herein indicated as underlined) wherein C 1228 of the wild type C57/BL6/J sequence is altered to A 1228, causing a codon change (CAG to AAG):
  • a nucleic acid sequence containing the complete coding sequence for the human wild type dudulin 2 gene is shown below with a putative SNP position indicated as underlined.
  • the dudulin 2 transcript presented below was obtained from public domain data (Accession No. XM_002477 . 3). We deduced the SNP position on the basis of the corresponding position in the mouse gene:
  • An embodiment of the present invention contains a mutated dudulin 2 gene; for example, wherein the coding sequence is that of the human dudulin 2, wherein C 1317 is altered to A 1317, causing a codon change (CAG to AAG):
  • a further embodiment of the present invention is a polynucleotide comprising the complete coding sequence for the human mutant dudulin 2 gene (as shown below with the location of the mutant SNP disclosed herein indicated as underlined) wherein C 1317 of the wild type C57/BL6/J sequence is altered to A 1317, causing a codon change (CAG to AAG) as shown below in SEQ ID NO:5. (Further mutations of the wild type sequence of the invention include: A 1318, or G 1319 not to A.)
  • Gin 395 (SEQ ID NO:8) is changed, e.g., to Lys 395 (SEQ ID NO:6).
  • Other point mutations in the same codon i.e., any base switch at position 1317 or 1318 ofthe wild type coding sequence, or any base switch ofthe wild type coding sequence but to A at 1319, would result in a non-silent mutation (encode an amino acid substitution).
  • Murine and human dudulin 2 are closely related as can be seen from the alignments shown in Figure 4 and 5. But both proteins are not only conserved on amino-acid level as can be seen from Figure 7.
  • Figure 7 A illustrates the conserved syntheny between mouse and man within the dudulin 2 region. The table was extracted from Ensemble (http://www.ensembl.org/Mus musculus on May 14 th 2002, using the "export data" function. A gene list for the mouse chromosomal region between 120 and 122 Mb was created giving the human orthologues. It can easily be seen that the order of genes is conserved between mouse and human. Furthermore, the human and murine genes show striking homologies concerning their exon-intron structure, as can be seen in Figure 7B.
  • exon sizes of coding sequences are exactly the same between mouse and human. Exon sizes of non coding sequences (underlined) are conserved in the 3'-region but not in the 5'-end. Intron-sizes (written below) show some variation, but in general the exon-intron structure is related.
  • dudulin 2 protein family consisting of 4 murin and 4 human orthologues.
  • STEAP six-transmembrane epithelial antigen of the prostate
  • TIARP TNF- ⁇ -induced adipose-related protein
  • Figure 5 shows an amino acid sequence alignment of all murine and human family members, done by CLUSTAL W(Astra Draco AB, Lund, Sweden: http://circinus.ebi.ac.uk:6543/cgi-bin/clustalw.cgi). It reveals the close relation of mouse and human orthologues as well as the relation of all family members with each other. The closest murine homologue to dudulin 2 is found in dudulin 3. The Gin (Q)- residue mutated in dudulin 2 is conserved throughout the whole family (highlighted in Fig. 5).
  • the ⁇ KOS-System a yeast-bacteria shuttle system (Wattler et al, 1999, Biotechniques, 26: 1150-1159) may be used to construct a recombinant vector to insert a point mutation at nucleotide 1228 in the mouse dudulin 2 gene according to well known techniques.
  • Oligonucleotides are used in PCR reactions to amplify two fragments, named A and B, respectively, which are cleaved by Sfi after purification by QIAquick kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and then ligated to a yeast/bacteria-selection cassette (Wattler et al, 1999, supra). Wild type yeast are then transformed with the ligation product (A + B + yeast/bacteria selection cassette) and the pKOS-clone together and a double selection is performed by plating onto defined growth medium lacking uracil and tryptophan.
  • yeast able to survive are those carrying a homologous recombination between the selection cassette with the ligated fragment A + B + yeast/bacteria selection cassette and the genomic pKOS-clone DNA.
  • Single yeast colonies were propagated and tested for the presence of the point mutation by colony-PCR with the oligonucleotide 5' and 3' primers, respectively.
  • ES-cell DNA is digested and hybridized with a 5 '-external probe. A single integration is verified by hybridization of the same Southern blot with a probe generated from the selection cassette.
  • Clones containing the point mutation were used to produce chimeric mice by blastocyst injection and transfer using standard methodology, well known in the art.
  • the chimeras are bred to wild type mice and in the following generation the same Southern strategy as outlined above is used to determine germline transmission. We generate traceable heterozygotes and then homozygotes according to well known techniques.
  • the PCR-Product was purified with the QIAquick PCR Purification Kit (Quiagen) according to the manufacturer's instructions.
  • the PCR-product was sequenced as previously described (Example 1, Item 5: DNA Sequencing) to exclude errors within the sequence.
  • the amplified sequence was found to be identical to the coding region of SEQ ID NO:7.
  • Entry clones were generated in vector pDONR201 using the Gateway-BP Cloning Kit according to the manufactures instructions (BP reaction).
  • DH5 ⁇ Competent Cells were transformed with l ⁇ l of the BP reaction following well known standard procedures. Selection was done on LB plates containing 50 ⁇ g/ml kanamycin. Plasmid-DNA of selected clones was isolated with the help of the Plasmid-DNA NucleoSpin Kit (Macherey-Nagel) according to the manufacturers instructions. Sequencing of Plasmid- DNA was done as described above.
  • DH5 ⁇ Competent Cells were transformed with 1 ⁇ l of the LR reaction following well known standard procedures. Selection was performed on LB plates containing 100 ⁇ g/ml ampicillin. Plasmid-DNA of selected clones was isolated and sequenced as described above.
  • the further procedure resembles the one described above (Example 6, Item A).
  • the corresponding SEQ IDs are SEQ ID NO:l for wild type murine dudulin 2 and SEQ ID NO: 3 for mutant murine dudulin 2.
  • n Dudulin2-HA For murine cDNAs as template: n Dudulin2-HA:
  • hDudulin2-HA 5'-ATGTACCCATACGATGTTCCAGATTACGCTCTTATGCCAGAAGAGATGGACAAG (SEQ ID NO:44)
  • hDudulin2-4 5'-GTGCCCGGGGTCCAGAGCC (SEQ ID NO.45)
  • the PCR-Product was purified with the QIAquick PCR Purification Kit (Quiagen) according to the manufactures instructions and sequenced as previously described. We then performed a second PCR-reaction with Taq-Polymerase (Sigma) according to the manufacturers instructions using the previously generated PCR-products as template and the following primers:
  • HA-attB 1 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAATGTACCCATACGATGTTCC (SEQ ID NO.46)
  • mDudulin2-attB2 5'-
  • HA-attBl (SEQ ID NO:46)
  • hDudulin2-attB2 5'- GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATACGTGGCTCGTCTTC (SEQ ID NO.48)
  • the further procedure resembles the one described above (Example 6, Item A).
  • the corresponding SEQ IDs are SEQ ID NO:l for wild type murine dudulin 2, SEQ ID NO: 3 for mutant murine dudulin 2 and SEQ ID NO: 7 for human dudulin 2.
  • cDNA 10 coding sequence (cDNA) of wild type dudulin 2 (mouse, C57/BL6 strain)

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Abstract

La présente invention concerne un modèle animal non humain, en particulier un modèle souris, de troubles hématologiques entraînés par l'expression d'une duduline 2 modifiée. L'invention se rapporte également à la duduline 2 et aux acides nucléiques codant cette dernière, de même qu'aux protéines et acides nucléiques duduline 2 de souris et humains recombinés modifiés correspondants. L'invention concerne en outre des utilisations du modèle animal non humain, des dudulines 2 modifiées et de leurs homologues dudulines 3, en particulier l'utilisation de la duduline 2 humaine modifiée dans le traitement d'états médicaux associés à une surexpression de la duduline 2.
PCT/EP2003/005920 2002-06-06 2003-06-05 Genes de la duduline 2, produits d'expression, modele animal non humain : utilisations dans les maladies hematologiques humaines WO2003104270A2 (fr)

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